LDL plays a role of supplying cholesterol to peripheral cells, and it is a direct factor for various types of arteriosclerosis including coronary arteriosclerotic disease as a typical example. An increase in LDL-C is a principal risk factor for arteriosclerotic diseases. Thus, fractional determination of such LDL-C is clinically useful.
Conventional methods for quantitatively determining LDL-C include an ultracentrifugation method, an electrophoresis, and a calculation method using a Friedewald formula.
The ultracentrifugation method is a method involving utilization of a difference in the gravities of lipoproteins, which comprises separating LDL with the use of an ultracentrifuge and then measuring the cholesterol amount (Non Patent Literature 1).
However, separation operations according to such an ultracentrifugation method are complicated, and thus, this method is disadvantageous in terms of promptness and simplicity.
The electrophoresis includes a method utilizing a difference in the charges of lipoproteins, which comprises separating LDL using an agarose gel as a supporting medium, and a method utilizing a difference in the particle sizes of lipoproteins, which comprises separating LDL using a polyacrylamide gel as a supporting medium. However, the electrophoresis is poor in terms of quantitative capability, and it is problematic in terms of simplicity, economic efficiency, etc.
In the calculation method using a Friedewald formula, based on the measurement values of total cholesterol (hereinafter abbreviated as T-C), cholesterol (hereinafter abbreviated as HDL-C) in high-density lipoprotein (hereinafter referred to as HDL), and total triglyceride (hereinafter abbreviated as T-TG), the amount of LDL-C is calculated according to the formula shown below (Non Patent Literature 2).(LDL-C)=(T-C)−(HDL-C)−(T-TG)/5
However, since this method is affected by the content of T-TG in the serum or diet, it has a problem in accuracy.
In recent years, there have been reported methods for quantitatively determining LDL-C, which can be applied to commonly used auto-analyzers, without requiring separation operations performed by the ultracentrifugation method and the like.
Among such methods, the following methods for quantitatively determining LDL-C are known.
A method for quantitatively determining LDL-C contained in a test sample, which comprises: a first step of allowing cholesterol esterase and cholesterol oxidase to act on a test sample in the presence of a surfactant acting on lipoproteins other than LDL, and then removing the formed hydrogen peroxide, to quench cholesterol in HDL, very low-density lipoprotein (hereinafter referred to as VLDL) and a chylomicron contained in the test sample; and a second step of quantitatively determining residual cholesterol contained in the sample (Patent Literature 1).
A method for quantitatively determining LDL-C, which comprises: adding a surfactant selected from a polyoxyethylene alkylene phenyl ether and a polyoxyethylene alkylene tribenzyl phenyl ether and an enzyme reagent for measuring cholesterol to the serum; reacting these reagents preferentially with cholesterols in HDL and VLDL among lipoproteins; and measuring the reaction amount of residual cholesterol (Patent Literature 2).
A method, which comprises adding a polyoxyethylene derivative, a polyoxyethylene-polyoxypropylene copolymer and enzymes for measuring cholesterol to a biological sample, and selectively measuring LDL-C among lipoproteins (Patent Literature 3).
A method for measuring LDL-C, which comprises measuring LDL-C contained in a biological sample in the presence of dimethyl-α-cyclodextrin and/or poly-β-cyclodextrin (Patent Literature 4).
A method for directly and selectively measuring cholesterol contained in a sample comprising at least one of chylomicron, HDL, LDL and VLDL, wherein LDL-C contained in the sample is quantitatively determined in the presence of a compound containing a phospholipid or a phospholipid-like group (Patent Literature 5).
However, it has been desired to develop a method and a kit for more simply and precisely measuring LDL-C contained in a sample.